Trans-resveratrol (trans-3,4′,5-trihydroxystilbene, FIG. 1), and derivatives such as piceid (Larronde et al., 2005; Rimando and Barney, 2005), along with pinosylvin (Celimene et al., 1999) belong to a class of naturally occurring defense compounds that are produced in a select number of plant species and known as stilbenes. These plant polyphenols are receiving considerable interest based upon a number of associated health benefits (Baur and Sinclair, 2006; Delmas et al., 2006). Most notably, the significant levels of the resveratrol metabolite in red wine have been credited to the phenomenon referred to as “the French Paradox”. It was observed in a large population study that prolonged, moderate consumption of red wine correlated with a very low incidence of cardiovascular disease (most notably coronary heart disease) among this study group despite a life-style that included a high saturated fat diet, little exercise and widespread smoking (Frankel et al., 1993; Kopp, 1998). Over the last decade, resveratrol has been reported to be associated with numerous other health benefits ranging from its function as a general anti-oxidant, to its anti-cancer, -atherosclerosis and -aging properties and most recently its neuroprotective and estrogenic activities (Gehm et al., 1997; Miura et al. 2003; Orallo, 2006). Furthermore several natural derivatives of resveratrol have shown additional health benefits including a methylated resveratrol compound, pterostilbene (FIG. 1), that has been shown to reduce cholesterol levels in laboratory animals (Rimando et al., 2005). Pinosylvin, another relative in the stilbene pathway has been associated with anti-inflammatory and cancer chemopreventative activities (Park et al., 2004).
With a growing trend in the United States and the continued popularity in Europe and Asia, for seeking natural health enhancing products, many plant-derived nutraceuticals are being incorporated into the functional food industry, the herbal and dietary supplement markets, and pharmaceutical industry. Countless studies have shown that US consumers often prefer foods with added health benefits over the same food without the benefit, and inclusion of these health-enhancing compounds in food products is preferred to taking dietary supplements. While dried or extracted plant material (seeds, roots, rhizomes, etc.) enriched in resveratrol and other stilbenes are incorporated into a number of marketed products that include dietary supplements (i.e. Longevinex™) and health-enhancing food products (i.e. Old Orchard Beverage Company, Sparta, Mich.), this source of resveratrol and other stilbenes is typically associated with color pigments and numerous other components that limit their broader application into food, nutritional and cosmetic products. A high quality source of naturally-derived resveratrol and its many derivatives that is void of color, taste, odor as well as production contaminants (i.e. pesticide residues, heavy metals, etc.) is currently not available on the commercial market due to a lack of consistent, high volume, cost-effective production systems for these health beneficial plant metabolites.
Efforts to advance production systems for providing more enriched and concentrated commercial stocks of resveratrol have taken several distinct strategies. The reconstruction of a biochemical pathway in a heterologous host to produce resveratrol was first demonstrated in wine yeasts with the intent of increasing resveratrol production for health benefits during fermentation in both red and white wines (Becker et al., 2003). More recent efforts have successfully co-expressed several genes belonging to the stilbene biosynthesis pathway of peanut in E. coli (Watts et al., 2006). While the conversion of the substrate 4-coumaric acid was functional in this recombinant microbial bioproduction system and produces over 50 times the levels of resveratrol than recombinant yeast (100 mg/L in E. coli), issues of inefficient substrate utilization, high substrate cost and recombinant-based production issues currently limit commercialization efforts of resveratrol product from these systems. In other attempts to produce resveratrol, genes encoding resveratrol were introduced into legume plant cells (Paiva et al., U.S. Pat. No. 6,974,895). Lengthy process steps and cost are among the disadvantages of such systems.
The use of a natural plant-based bioproduction approach for producing this plant-derived resveratrol has several advantages. While the use of grapevine cell suspensions for the production of trans-resveratrol has reported levels as high as 15 mM in the spent medium (Bru et al., 2006), there are issues surrounding long-term stability of plant cell cultures for secondary metabolite production (Wink et al., 2005). Such cultures are undifferentiated and in order to maintain the cultures ongoing hormone exposure is required, and stability becomes a problem. The culture can stop producing the stilbene and not respond to elicitors.
Accordingly, there exists a need to improve on systems for controlled, contained production of enriched fractions of natural stilbenoids that include resveratrol, pinosylvin and their respective derivatives.